Farmaceutische, Biomedische en Dier­geneeskundige Wetenschappen

Doctoraten 2021

Departement Biomedische Wetenschappen

Systematische beeld-gebaseerde profilering van neuronale connectiviteit. Van de synaps tot de celkern. - Marlies Verschuuren (28/06/2021)

Marlies Verschuuren

  • 28 juni 2021, 16u30 - 18u30
  • Online verdediging
  • Promotor: Winnok De Vos

Abstract

Long-term adaptations of the brain, including learning, memory formation, addiction and chronic pain sensation, rely on a delicate balance of tunable connections between neurons. In numerous neurological diseases this neuronal connectivity is perturbed. Neuronal connectivity is classically assessed by means of morphological correlates such as the density of synapses or functional readouts such as synchronous calcium spiking activity. However, such approaches operate with blinders as they do not account for multifaceted effects. Furthermore, recent reports have inferred an important role for the nucleus in neuronal connectivity. As yet, nuclear morphology has not been regarded as a potential readout of neuronal connectivity and limited tools are available to capture it with sufficient sensitivity.

We established a detailed interrogation of morphological and functional correlates of neuronal connectivity in one single microscopy-based assay to screen for its modulators in primary neuronal cultures. The integrative approach proved to be more sensitive than more classical single-readout approaches. Using this pipeline, we found that inhibition of dual leucine zipper kinase (DLK) increased neuronal connectivity in primary cortical cultures, albeit within a specific time and dose range. This neuroprotective effect was not only observed in basal conditions but also in cultures depleted from antioxidants and in cultures in which microtubule stability was genetically perturbed, suggesting it has broad-spectrum therapeutic relevance. In addition to this integrated assessment of neuronal connectivity, we optimized an image analysis algorithm that accurately detects nuclei in fluorescence images and captures their morphology by precise delineation of their contour and by extraction of robust, quantitative descriptors.

Having established a method to interrogate nuclear morphology and to connect it with neuronal connectivity, we next sought to evaluate the influence of targeted perturbations to nuclear morphology. Given their crucial role in nuclear architecture and function, we thereby focused on the nuclear lamins. A significant decrease in lamin B1 levels with culture time and brain aging, drove us to interrogate the effect of its selective depletion in human neuroblastoma model cells using shotgun proteomics. In-depth analysis revealed that lamin B1 acts as a molecular switch controlling differentiation, by tempering integrated stress levels.

The results that have been generated in this thesis, illustrate that profiling microscopy images with deep coverage enables sensitive interrogation of neuronal connectivity and allows exposing a pharmacological window for targeted treatments. Our work also proves that nuclear lamins have a crucial role in steering neuronal cell function and may be considered as a putative target for novel modulators of neuronal connectivity.

Biomerkers van maladaptieve plasticiteit tijdens epileptogenese met in vivo hersenbeeldvorming - Daniele Bertoglio (28/06/2021)

Daniele Bertoglio

  • 28 juni 2021, 18u - 20u
  • Online verdediging / Promotiezaal, CDE (voor beperkt publiek, na registratie bij student)
  • Promotoren: Annemie Van der Linden, Jeroen Verhaeghe, Stefanie Dedeurwaerdere

Abstract

​Epilepsy is one of the most common chronic neurological conditions affecting more than 65 million people worldwide. It may be genetic or acquired following different types of brain injury, such as status epilepticus (SE), trauma, or stroke. Temporal lobe epilepsy (TLE) represents the most common form of drug-resistant focal epilepsy in adults. In TLE, the initial brain insult is followed by a latent period during which several pathophysiological changes occur, including brain inflammation, impairment of networks, and synaptic circuit alterations. Ultimately, these modifications result in the transition of a healthy brain into a hyperexcitable one leading to the development of spontaneous recurrent seizures. This process is defined as epileptogenesis. 

The seizure-free period lasting between the epileptogenic insult and the clinical onset represents a therapeutic window of opportunity for disease-modifying therapies aimed at halting or preventing the process of epileptogenesis. Despite the attractive therapeutic paradigm, the development of (pre)clinical studies to investigate the efficacy of antiepileptogenic treatments is severely hampered by the wide heterogeneity in insults and our inability to predict and stratify subjects at risk. 

This thesis aims to characterize novel clinically relevant in vivo imaging markers of epileptogenesis in the kainic acid-induced status epilepticus (KASE) rat model of TLE to identify prognostic markers for the long-term disease outcome. Specifically, using positron emission tomography (PET) and magnetic resonance imaging (MRI), we focused on the assessment of translocator protein 18kDa (TSPO) upregulation as a proxy for neuroinflammation as well as functional connectivity changes as a read-out for the epileptogenic neuroplasticity. 

First, we compared the KASE rat model of TLE to select the most appropriate strain to elucidate pathological changes during epileptogenesis given the divergences in disease progression and seizure outcome. Secondly, we demonstrated that a single TSPO PET scan at disease onset (2 weeks post-SE) could accurately predict the seizure frequency of each epileptic rat, suggesting its potential prognostic value for the stratification of subjects at risk, differentiating categories of epileptic rats with significantly distinct seizure burden. Lastly, by investigating functional network connectivity changes during epileptogenesis, we observed a widespread network hyposynchrony at disease onset as well as diverging changes depending on disease severity. 

In conclusion, the work of this thesis identified relevant preclinical prognostic imaging markers of epileptogenesis predictive of seizure frequency. Future studies will be necessary to understand whether these markers might be utilized clinically.

Verbetering van de diagnose van rifampicine-resistente tuberculose: programmatische aspecten, diagnostische uitdagingen, en moleculaire epidemiologie - Semuto Jean Claude Ngabonziza (23/06/2021)

Semuto Jean Claude Ngabonziza

  • 23 juni 2021, 16u - 18u
  • Online verdediging: https://Itg.zoom.us/j/85897541694?pwd=K0RtUlh2aGlQOVhLQnlKazAxaWxuUT09
  • Promotoren: Leen Rigouts, Bouke de Jong
  • Abstract

    Tuberculosis (TB) remains the leading infectious disease cause of mortality globally. Resistance to rifampicin (i.e. the most powerful anti-TB drug) impedes optimal management of patients and TB control. Indeed, the majority of rifampicin-resistant (RR) TB patients remain undiagnosed and untreated, thus spreading resistant Mycobacterium tuberculosis complex (MTB) strains. In Rwanda  the first RR-TB patients were diagnosed in 1989. However,  adequate programmatic management of RR-TB started 15 years later, in response to an increasing prevalence and poor treatment outcomes. Stepwise interventions were implemented aiming at early diagnosis and appropriate treatment. First, the program expanded access to culture-based RR-TB detection. Second, rapid molecular tests, such as Xpert MTB/RIF, were implemented to swiftly diagnose RR-TB. My Ph.D. research showed a substantial reduction of delays in initiating RR-TB treatment from 175 days in 2006 to 5 days only in 2016. This reduction of delays decreased RR-TB related mortality from 30.8% in 2006 to 6.9% in 2016. Besides reduced mortality, I showed that such swift detection interrupted the spread of RR-TB, also that most RR-TB in Rwanda was caused by a single MTB clone that we named ‘Rwanda rifampicin-resistant TB clone’, or ‘R3clone’. Through universal access to rapid rifampicin resistance testing, the R3clone population declined since 2014. However, the extensive use of Xpert MTB/RIF did not only yield positive effects. Ten years after its global rollout, my analysis showed very important pitfalls of Xpert MTB/RIF. As more and more patients were tested with Xpert MTB/RIF, a substantial proportion of patients were diagnosed early with paucibacillary disease. I showed that half of these patients diagnosed with RR-TB in fact had rifampicin-susceptible TB. The Xpert MTB/RIF software erroneously interprets insufficient DNA binding as evidence of resistance. Unfortunately, these patients were unnecessarily treated with a longer ‘second line’ treatment regimen with more toxic drugs. Based on my findings the National TB Programme in Rwanda changed the diagnostic algorithm, to further ascertain RR in patients with a paucibacillary sample before starting RR-TB treatment. Today, TB patients in Rwanda only receive second-line treatment if they really need it. Besides the challenges in diagnosing RR-TB, I identified a novel MTB lineage that we named lineage 8 (L8). The L8 is a sister clade to the known MTB lineages. Remarkably, the two L8 strains identified so far were resistant to key anti-TB drugs. L8 seems to be extremely rare and restricted to the Great Lakes region.

Ontwikkeling van efficiënte therapieën voor vasculaire media calcificaties, een belangrijk klinisch probleem in onze vergrijzende maatschappij - Britt Opdebeeck (31/05/2021)

Britt Opdebeeck

  • 31 mei 2021, 16u30 - 18u30
  • Promotiezaal (CDE) of ONLINE
  • Promotoren: Patrick D'Haese, Anja Verhulst

Abstract

Arterial media calcification or the deposition of calcium-phosphate crystals (i.e. hydroxyapatite) in the medial layer of the arterial wall is a major cardiovascular complication in elderly and patients with chronic kidney disease (CKD), diabetes and osteoporosis. This pathology favors arterial stiffness, hypertension and left ventricular hypertrophy ultimately leading to heart failure, impaired coronary perfusion and cardiac stroke. Moreover, CKD patients with known arterial calcification have to be considered at highest cardiovascular risk. As no effective treatment strategy is available at this time, finding novel and efficient therapies that directly target the mineralization process in the vessel wall are urgently needed. The first research objective focused on the role of protein-bound uremic toxins in the arterial media calcification process. This objective revealed that, in a context of CKD, protein-bound uremic toxins, indoxyl sulfate and p-cresyl sulfate, directly promoted arterial media calcification via activation of inflammation and coagulation pathways and were strongly associated with impaired glucose homeostasis. The second research objective investigated the role of extracellular nucleotides in arterial media calcification through activation of purinergic independent (tissue-nonspecific alkaline phosphatase (TNAP) inhibition) and dependent (P2X1 receptor) pathways. The enzyme TNAP mediates the hydrolysis of calcification inhibitor pyrophosphate (a metabolic degradation product of nucleotides) into the calcification stimulator inorganic phosphate. We found that a dosage of 10 mg/kg/day TNAP-inhibitor SBI-425 was sufficient to inhibit the development of mild arterial media calcification (i.e. warfarin rat model) however, turned out to be insufficient for treating moderate to severe CKD-related arterial media calcification (i.e. adenine rat model). Also treatment with TNAP-inhibitor SBI-425 resulted into bone mineralization side-effects. Similarly, P2X1 receptor agonist b,y-meATP prevented the development of mild arterial media calcification (i.e. warfarin rat model) however provoked also deleterious effects on bone mineralization. Since patient populations developing arterial media calcification (CKD, diabetes as well as osteoporosis patients) already suffer from a seriously compromised bone health this is of significant clinical importance. Therefore, exploring new therapeutic targets related to inflammation, coagulation and/or glucose homeostasis, investigating the dissimilarities between the calcification process in the arteries and bone, as well as trying to selectively target compounds such as β,γ-meATP to the blood vessels by using i.e. nanoparticle conjugated to antibody drug delivery system might all be promising ways to obtain safe and efficient anti-arterial calcification therapies.

Een proteoom gebaseerde beschrijving van de Withaferin A antitumor eigenschappen - Martin Dom (12/03/2021)

Martin Dom

  • 12 maart 2021, 16u - 18u
  • Online verdediging
  • Promotoren: Xaveer Van Ostade, Wim Vanden Berghe

Abstract

Ondanks de veelbelovende multifunctionele antitumor eigenschappen van Withaferine A (WA), ontbreekt een algemeen overzicht van de onderliggende moleculaire antitumor mechanismen. Dit zet een rem op het potentieel klinisch gebruik van WA. Multifunctionele chemotherapie is een veelbelovende strategie in het bestrijden van multidrug resistente tumoren en we wilden met deze thesis de kennis leegte tussen WA behandeling en de geobserveerde kanker celdood opvullen.

Om deze leemte in kennis op te vullen, hebben we een kwantitatieve chemoproteomics workflow ontwikkeld waarmee we de eiwit bindingspartners van WA hebben gekarakteriseerd. Deze data, gecombineerd met differentiële eiwit expressie data, hebben ons tot de hypothese geleid dat WA een acute stress induceerder is én tegelijk de stress homeostasis response als doelwit heeft. Hiertoe behoren o.a. de oxidatieve stress response, het ubiquitine-proteasome systeem, autofagie en ERAD response. Zelfs wanneer kanker cellen de basale cellulaire stress levels herstellen, dan beïnvloedt WA ook meer structurele, non-stress, cellulaire events. Hiertoe behoren o.a. cytoskelet verzwakking, onderbreken van de cel cyclus en van de Akt/NFkB pathways. Verder identificeerden we een nieuwe manier van WA proteasome inhibitie die verloopt via de binding met de deubiquitinerende enzymes USP14 en UCHL5.

We geloven dat onze resultaten aantonen dat de technologische vooruitgang ons toelaat om target-based en observatiegebaseerde drug ontwikkeling strategiën te combineren en zo een heel nieuw veld van antitumorale therapiën te verkennen.

Strategieën voor het efficiënt opnemen en reconstrueren van structurele en quantitatieve MR beelden - Maarten Naeyaert (11/02/2021)

Maarten Naeyaert

  • 11 februari 2021, 17u - 19u
  • Online verdediging
  • Promotoren: Marleen Verhoye, Jan Sijbers

Abstract

Magnetic resonance imaging (MRI) is a valuable tool for investigating the brain, both in the clinic and in research. Its main drawback is its long acquisition time, which can be shortened by minimising the amount of data which is acquired and by using it efficiently. Three objectives were set for this thesis, each of which makes efficient use of the acquired data.

Two objectives relate to compressed sensing (CS). CS algorithms allow the reconstruction of signals or images from incomplete data, by acquiring this data in a pseudo-random way and finding the most compressible image still consistent with the acquired data. The final image quality depends on which data is sampled, and several acquisition strategies were tested on both simulated signals, test objects, and using actual acquisitions. Firstly, CS was implemented for a fast spin echo (FSE) sequence, and it was found that optimal results occur when the majority of the data is sampled in the centre of k-space, with progressively less data being sampled closer to the edge of k-space.

Secondly, a method to accelerate the acquisition of isotropic in-vivo high-angular radial diffusion imaging (HARDI) data using an FSE sequence was developed. While slow, the FSE sequence results in few artefacts, and CS can be used to acquire the large number (>50) of volumes required for HARDI faster. In this case, CS can also be used to subsample the diffusion signal, in the q-space, to acquire fewer volumes. Using knowledge from the first objective, strategies for subsampling the data, either only in q-space or both in k-space and q-space, were tested. We found that subsampling the q-space only is most efficient, and 15 to 20 volumes proved sufficient to yield high quality reconstructions without major differences in quantitative diffusion measures or tractography results.

Finally, a method was developed to quantify micron-sized iron-oxide particles (MPIO) in MRI-images. These particles are used to label cells and cause a negative contrast, i.e. hypo-intensities. However, positive contrast images can be constructed to improve the visibility and localisation of MPIO, which was done efficiently from a single acquisition. To quantify MPIO robustly and reliably the normalized average range (nAR) is introduced. The nAR compares the average value of regions of interest (ROI’s) to that of a control ROI in range filtered images, and showed a higher sensitivity in optimized positive contrast images and is not sensitive to the bias field of the receiver coil.

Verdere inzichten in de pathogenetische mechanismen van melorheostose en de ziekte van Paget - Raphaël De Ridder (26/01/2021)

Raphaël De Ridder

  • 26 januari 2021, 16u30 - 18u30
  • Online verdediging
  • Promotoren: Wim Van Hul, Geert Mortier

Abstract

Dysregulation of bone remodeling can lead to a large number of skeletal disorders. Elucidating the molecular background of such pathologies has the potential of improving diagnosis and can be of great value in the identification of novel targets for the development of novel therapies. The general aim of this thesis was therefore to investigate the contribution of genetic factors in the pathogenesis of two bone disorders, melorheostosis and Paget’s disease of bone (PDB).

In a first part, we performed a mutation analysis in a small cohort of melorheostosis cases. This is a very rare condition characterized by asymmetric increased density of the cortical bone and alteration in surrounding soft tissues. This analysis confirmed the importance of somatic pathogenic variation in the negative regulatory region of MAP2K1 and showed the presence of a novel melorheostosis-associated somatic p.Cys121Ser variant in the catalytic core. Gene-set enrichment of transcriptomic data indicates that this variant leads to hyperactivation of proliferative pERK signaling. 

The second part of this thesis was focused on PDB. Follow-up of families with a history of PDB further supports the importance of genetic variation in SQSTM1. Based on the molecular background of a number of PDB-related phenotypes, targeted sequencing was performed for a panel of 52 candidate genes. This indicated a significant association for variation in the RIN3 gene with the phenotype, with the rs117068593 variant conferring a protective effect that modifies the age of onset in patients. A panel-wide gene burden analysis highlighted association for a number of genes (TNFRSF11A, NUP205, VCP and NFKBIA) with PDB pathogenesis and suggested an enrichment of rare genetic variation in several other genes (NR4A1, NUP205 and PRKCI). Finally, the use of next-generation sequencing technologies is rapidly expanding the number of candidate genes that requires further functional evaluation. Therefore, we generated a loss-of-function model of the ubiquitin-associated domain in the sqstm1 gene of zebrafish. Preliminary results support the presence of a skeletal phenotype in our model. Further studies will be needed to elucidate whether this reflects an osteoclast-driven Pagetic phenotype.

Together, our results provide further insights into the regulation of bone remodeling and explore a novel platform to study the pathogenesis of skeletal disease in vivo.

Drug-resistentie tijdens viscerale leishmaniasis: impact op de infectiviteit van de parasiet in de gewervelde gastheer en zandvlieg vector - Dimitri Bulté (26/01/2021)

Dimitri Bulté

  • 26 januari 2021, 16u - 18u
  • Online verdediging
  • Promotoren: Guy Caljon, Louis Maes

Abstract

Visceral leishmaniasis (VL) is causing 50,000 to 90,000 new cases annually and more than 20,000 deaths which makes it the deadliest parasitic disease after malaria. In the absence of a successful vaccine, the main VL control strategy is dependent on chemotherapy, however, only a few antileishmanial treatment options are available. Miltefosine (MIL) is currently the only oral drug for VL but is failing to fully clear parasites in an increasing number of patients. These treatment failures could initially not be linked to the emergence of resistance, although more recently a few MIL-resistant (MIL-R) clinical isolates have been described. In order to safeguard the use of MIL for future VL therapies, it is essential to evaluate the impact of MIL-resistance on parasite fitness in the vertebrate host and sand fly vector as this could indicate the potential spread of resistant parasites into the population.

In accordance with previous studies using a laboratory MIL-R L.infantum, a decreased infectivity of the vertebrate host was observed for a natural MIL-R L. infantum. However, no impact on the development of the parasite in the sand fly vector was observed. These results indicate that the impact of resistance on parasite fitness in the vertebrate host and insect vector may not necessarily be the same and indicate the potential transmission of MIL-R parasites. To enable a combined study of the infection dynamics and underlying immunological events for differential in vivo infectivity and drug efficacy, firefly luciferase (PpyRE9) / red fluorescent protein (DsRed) double-reporter strains were generated of laboratory MIL-R and syngeneic MIL-sensitive (MIL-S) Leishmania infantum. Results show that MIL-R parasites induce an increased innate immune response that is characterized by enhanced influx and infection of neutrophils, monocytes and dendritic cells in the liver and elevated serum IFN-γ levels, finally resulting in a relatively lower burden of MIL-R parasites in liver macrophages. The elevated IFN-γ levels were shown to originate from an increased response of hepatic NK and NKT cells to the MIL-R parasites which contributed to the attenuated MIL-R phenotype. In addition, it was demonstrated that the presence of MIL could increase the in vivo fitness of MIL-R parasites by lowering NK and NKT cell activation, leading to a reduced IFN-γ production. These results highlight the potential risk of MIL treatment in sustaining infections with resistant parasites. Close monitoring of parasite drug susceptibility and adjusted treatment protocols would therefore be beneficial.